Conventionally, a variety of biosensors have been proposed as a system for simply quantifying a specific component in a sample solution without diluting or stirring the sample solution. As one example of the biosensors, for instance, the following sensor has been known (Japanese Laid-Open Patent Publication No. Hei 2-062952).
This biosensor is fabricated by forming an electrode system comprising a working electrode, a counter electrode and a reference electrode on an electrically insulating base plate by screen printing or other method and forming thereon an enzyme reaction layer comprising a hydrophilic polymer, an oxidoreductase and an electron acceptor in contact with the electrode system.
When a sample solution containing a substrate is dropped on the enzyme reaction layer of this biosensor, the enzyme reaction layer is dissolved, and the substrate and the enzyme react with each other, thereby reducing the electron acceptor. Thereafter, the reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate in the sample solution can be determined from an oxidation current value obtained in this oxidation.
According to the biosensor as mentioned above, in theory, it is possible to measure various substances by selecting an enzyme whose substrate is a substance to be measured.
For instance, if glucose oxidase is selected as the enzyme, it is possible to fabricate a glucose sensor for measuring the concentration of glucose in a sample solution.
In the biosensor having the structure as mentioned above, the enzyme is normally retained in the sensor in a dried state. Since the enzyme is composed mainly of protein, if the enzyme is exposed to moisture in the air, etc. over a long period, there is a risk of the denaturation of the enzyme. Moreover, in an extreme case, there is a risk of the inactivation of the enzyme.
For this reason, if the sensor is stored for a long time, the enzyme activity is lowered and the amount of enzyme that reacts with the substrate becomes insufficient, and thus there is a possibility that the resultant response current value is not proportional to the concentration of the substrate.
Therefore, in order to obtain a biosensor excelling in the storage stability, it is important to provide an environment for retaining the activity of the enzyme for a long time in the vicinity of the enzyme. Moreover, it is necessary to improve the response of the sensor by facilitating smooth movement of the electrons and substrate during an enzyme reaction.
On the other hand, in order to fabricate a high-performance glucose sensor, pyrrolo-quinoline quinone dependent glucose dehydrogenase (hereinafter referred to as the “PQQ-GDH”) is used as the enzyme. In the glucose sensor using the PQQ-GDH, since oxygen is not involved in the catalytic reaction of the PQQ-GDH, this sensor has a characteristic that the enzyme reaction does not receive any effect of dissolved oxygen in blood, etc. Therefore, the measurement value given by this glucose sensor never varies depending on the oxygen partial pressure in the sample solution. In other words, it is possible to obtain a high-performance sensor.
However, in the case where the PQQ-GDH is used as the enzyme of the glucose sensor, it has been revealed that there is a problem that the response value is lowered by storage. This means that the response value is lowered as the period of storage of the glucose sensor is longer. It is impossible to always use the sensor at a certain time after the fabrication of the sensor. Hence, with a sensor whose response value will be lowered by storage, it is impossible to accurately quantify the concentration of glucose.
In view of such problems, it is an object of the present invention to provide a high-performance glucose sensor having excellent storage stability and an improved response characteristic in an initial stage.